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Fusobacterium necrophorum, a Gram-negative, anaerobic bacterium, is an opportunistic animal and human pathogen that causes a variety of infections termed necrobacillosis. There are 2 subspecies of F. necrophorum (subsp. necrophorum and subsp. funduliforme) that differ morphologically and biochemically and in virulence. Leukotoxin, a secreted protein, is considered to be the major virulence factor. In camelids, F. necrophorum causes a variety of infections, generally involving the lips, tongue, pharynx, interdigital spaces, foot pad, larynx, mandible, or maxillary bones. The objective of the current study was to characterize the presumptive Fusobacterium isolates from a variety of necrotic infections in llama (Lama glama) and alpaca (Vicugna pacos) and determine whether the strains possess leukotoxin activities. A total of 7 isolates from alpaca and 2 isolates from llama were characterized. Based on growth characteristics in broth culture, and biochemical and polymerase chain reaction analyses, all 9 isolates belonged to subsp. necrophorum and possessed the putative hemagglutinin gene. Western blot analysis with antileukotoxin antibodies raised in rabbit showed the presence of leukotoxin protein in the culture supernatant of all isolates. Furthermore, flow cytometry of the culture supernatants demonstrated cytotoxicity to bovine and alpaca polymorphonuclear leukocytes (PMNs). The extent of cytotoxicity to either alpaca or bovine PMNs differed among camelid strains. The cytotoxicity of many of the camelid strains was higher (P < 0.05) toward alpaca PMNs compared to bovine PMNs. Fusobacterium necrophorum isolates from llama and alpaca are similar to bovine isolates, and leukotoxin may be a major virulence factor.
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Journal of Veterinary Diagnostic Investigation
25(4) 502 –507
© 2013 The Author(s)
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DOI: 10.1177/1040638713491407
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Brief Research Reports
Fusobacterium necrophorum is a Gram-negative, anaerobic,
and rod-shaped bacterium that causes a variety of suppura-
tive and necrotic infections, generally called necrobacillosis,
in animals and human beings.
9
Two subspecies of F. nec-
rophorum are recognized.
12
The subsp. necrophorum is more
frequently encountered in animal infections,
6
while clinical
isolates from human infections have the characteristics of
subsp. funduliforme.
13
The subsp. necrophorum is more viru-
lent
6
because of the presence and/or increased production of
potent virulence factors, such as leukotoxin, lipopolysaccha-
ride, and hemagglutinin.
9
Of these factors, leukotoxin is
considered to be the major virulence determinant in the
pathogenesis of animal infections.
9
In wild and captive animals, necrobacillosis has been
reported to cause infections of the oral cavity in white-tailed
deer (Odocoileus virginianus), mule deer (Odocoileus
hemionus), pronghorn antelopes (Antilocapra americana),
and blue duikers (Philantomba monticola; syn. Cephalophus
monticola fusicolor).
2,3,11,16
In camelids, F. necrophorum
infections occur on the lips, tongue, pharynx, interdigital
spaces, foot pad, larynx, mandible, or maxillary bones.
5
In
alpaca (Vicugna pacos), the organism has been reported to
cause osteomyelitis of mandibles and many disseminated
necrotic lesions.
5
Occasionally, the infection may be aspi-
rated to the lung, causing severe necrotizing pneumonia,
while infections in the interdigital space and foot pad can
lead to lameness.
5
The current study examined presumptive
Fusobacterium isolates from a variety of necrotic infections
in llama (Lama glama) and alpaca to identify the species and
subspecies, and to determine whether the strains possess leu-
kotoxin activity.
A total of 9 isolates, 2 from llama (61557 and 69521) and
7 from alpaca (11034, 61715, 68690, 68699, 73556, 118610,
and 1012060) were included in the study (Table 1). Fusobac-
terium necrophorum subsp. necrophorum strain A25 and
subsp. funduliforme, strain B35, both previously isolated
from liver abscesses of cattle,
6
served as reference cultures in
all the tests conducted to characterize the llama and alpaca
isolates. All strains were cultured in brain-heart infusion broth
a
(BHI) that was prereduced with 0.05% cysteine hydrochlo-
ride and anaerobically sterilized (PRAS).
15
The growth char-
acteristic (sedimentation or no sedimentation) of the isolates
491407JVD
XXX10.1177/1040638713491407Fusobacterium necrophorum from llama and alpacaKumar et al.
research-article2013
From the Departments of Diagnostic Medicine/Pathobiology (Kumar,
Amachawadi, Nagaraja, Narayanan) and Clinical Sciences (Anderson),
College of Veterinary Medicine, Kansas State University, Manhattan, KS.
1
Corresponding Author: Sanjeev Narayanan, K-246, Mosier Hall,
Department of Diagnostic Medicine and Pathobiology, Kansas State
University, 1800 Denison Avenue, Manhattan, KS 66506.
sanjeev@vet.k-state.edu.
Characterization of Fusobacterium
necrophorum isolated from llama and alpaca
Amit Kumar, David Anderson, Raghavendra G. Amachawadi,
Tiruvoor G. Nagaraja, Sanjeev K. Narayanan
1
Abstract. Fusobacterium necrophorum, a Gram-negative, anaerobic bacterium, is an opportunistic animal and human
pathogen that causes a variety of infections termed necrobacillosis. There are 2 subspecies of F. necrophorum (subsp.
necrophorum and subsp. funduliforme) that differ morphologically and biochemically and in virulence. Leukotoxin, a secreted
protein, is considered to be the major virulence factor. In camelids, F. necrophorum causes a variety of infections, generally
involving the lips, tongue, pharynx, interdigital spaces, foot pad, larynx, mandible, or maxillary bones. The objective of the
current study was to characterize the presumptive Fusobacterium isolates from a variety of necrotic infections in llama (Lama
glama) and alpaca (Vicugna pacos) and determine whether the strains possess leukotoxin activities. A total of 7 isolates from
alpaca and 2 isolates from llama were characterized. Based on growth characteristics in broth culture, and biochemical and
polymerase chain reaction analyses, all 9 isolates belonged to subsp. necrophorum and possessed the putative hemagglutinin
gene. Western blot analysis with antileukotoxin antibodies raised in rabbit showed the presence of leukotoxin protein in
the culture supernatant of all isolates. Furthermore, flow cytometry of the culture supernatants demonstrated cytotoxicity to
bovine and alpaca polymorphonuclear leukocytes (PMNs). The extent of cytotoxicity to either alpaca or bovine PMNs differed
among camelid strains. The cytotoxicity of many of the camelid strains was higher (P < 0.05) toward alpaca PMNs compared
to bovine PMNs. Fusobacterium necrophorum isolates from llama and alpaca are similar to bovine isolates, and leukotoxin
may be a major virulence factor.
Key words: Alpacas; Fusobacterium necrophorum subsp. necrophorum; leukotoxin; llamas.
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Fusobacterium necrophorum from llama and alpaca
503
in the PRAS-BHI broth was recorded, and the isolates were
tested biochemically using a commercial kit.
b
All isolates showed uniform turbidity in the broth cultures
(no sedimentation) and, based on the commercial identifica-
tion kit, all isolates were positive for indole production and
alkaline phosphatase activity, similar to subsp. necrophorum,
strain A25. Many of the isolates, except oral swab (73556)
and tongue abscess (1012060) isolates, obtained from llama
and alpaca were associated with infections involving the
bone. The alkaline phosphatase activity may help the organ-
ism in bone resorption creating a deep wound, and thus mak-
ing a more anaerobic environment for the bacterial growth.
17
The frequent association of F. necrophorum in camelids with
necrotic infections in bone needs further study and explanation.
To identify the organisms at the molecular level, different
polymerase chain reaction (PCR) assays were carried out
using a PCR machine.
c
The genus-specific PCR assay based
on the 16S ribosomal RNA gene
8
yielded an amplicon of 610
bp, confirming that all isolates belonged to genus Fusobacte-
rium (Fig. 1A). The subspeciation of the isolates was based
on PCR amplifications of hemagglutinin (haem) gene
1
and
the leukotoxin (lkt) operon promoter–containing intergenic
region.
18
The primer sequences, PCR running conditions,
and amplicon sizes are shown in Table 2. The PCR specific
for the hemagglutinin gene amplified a 315-bp product from
all llama and alpaca isolates and in the bovine strain of subsp.
necrophorum, but not in the bovine strain of subsp. funduli-
forme (Fig. 1B). The PCR assay designed to amplify the lkt
promoter–containing intergenic region of the subsp. nec-
rophorum amplified a product of 571 bp with all the isolates
and that of the bovine strain of subsp. necrophorum (Fig. 1C).
In contrast, the PCR assay designed to amplify the lkt
promoter–containing intergenic region of subsp. funduli-
forme gave a negative result with all of the camelid isolates
(Fig. 1D) and, as expected, amplified a product of 337 bp
with the bovine strain of subsp. funduliforme. A summary of
the PCR results is given in Table 1. Overall, the results
revealed that the Fusobacterium isolates from alpaca and
llama belonged to subsp. necrophorum. This is similar to a
previous observation that necrotic infections caused by
F. necrophorum in cattle are more often caused by subsp.
necrophorum.
9
The production of leukotoxin was tested by a Western blot
assay for the leukotoxin protein in culture supernatant. An
aliquot of each supernatant, containing 20 µg of protein as
determined by Bradford assay,
d
was loaded into each lane.
Rabbit polyclonal antiserum, raised against affinity-purified
leukotoxin from a bovine strain of subsp. necrophorum, was
used as the primary antibody
10
followed by incubation with
goat antirabbit immunoglobulin G conjugated with alkaline
phosphatase
e
as the secondary antibody. The culture superna-
tant was prepared by growing the isolates in PRAS-BHI
broth to an absorbance of 0.60–0.65 at 600 nm, pelleting
cells by centrifugation, filtering through a 0.22-µm filter,
f
and concentrating 60-fold with a 100-kDa molecular mass
cutoff filter.
f
The Western blot with concentrated superna-
tants from all strains showed 110 and 90 kDa bands, similar
to that of bovine strain of subsp. necrophorum A25 (Fig. 2).
A 250-kDa band was observed in strains 1012060, 61715,
69521, and 73556, but the band was absent in 11034, 68690,
68699, and 118610 strains and was faint in the bovine strain
of A25. The 150-kDa band was present in all strains except
in strains 11034, 68690, 68699, and 118610. Bands of 130
and 125 kDa were present in all strains, including the bovine
Table 1. Source of Fusobacterium necrophorum isolates from llama and alpaca and polymerase chain reaction (PCR) analysis results.
Leukotoxin gene promoter region PCR
specific for:
Animal/source Strain
Fusobacterium-
specific PCR
Hemagglutinin gene
(haem) PCR subsp. necrophorum subsp. funduliforme
Bovine
Liver abscess A25 + + +
B35 + +
Llama
Sequestrum of hind limb 61557 + + +
69521 + + +
Alpaca
Necrotic mandible 11034 + + +
Necrotic infection in maxillary bone 61715 + + +
of forelimb 68690 + + +
Tooth abscess, osteomyelitis of
mandible
68699 + + +
Oral swab 73556 + + +
Bone sequestrum 118610 + + +
Tongue abscess 1012060 + + +
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Kumar et al.
504
Figure 1. Polymerase chain reaction amplification of genes targeting Fusobacterium genus–specific 16S ribosomal DNA (A),
hemagglutinin (haem) gene (B), and leukotoxin (lkt) gene promoter specific for subsp. necrophorum (C) and for subsp. funduliforme
(D). Lane M: 100-bp DNA ladder; each lane (lanes 1–11) represents DNA template of different isolates. Lanes 1 (69521) and 2 (61557):
llama strains; lanes 3–9 (68699, 61715, 68690, 11034, 73556, 1012060, and 118610): alpaca strains; lanes 10 (subsp. necrophorum strain
A25) and 11 (subsp. funduliforme strain B35): bovine strains.
Table 2. Primers, target genes, polymerase chain reaction (PCR) running conditions, and amplicon sizes.
Primer Target PCR conditions
Product size
(base pairs) Reference
Fuso 1 (for): 5’-GAGAGAGCTTTGCGTCC-3’
Fuso2 (rev): 5’-TGGGCGCTGAGGTTCGAC-3’
Genus-specific 16S
ribosomal DNA for
Fusobacterium
94°C for 5 min; 35
cycles of 94°C for 30
sec, 60°C for 30 sec,
and 72°C for 30 sec;
and final extension at
72°C for 7 min
610 8
Haem (for):
5’-CATTGGGTTGGATAACGACTCCTAC-3’
Haem (rev):
5’-CAATTCTTTGTCTAAGATGGAAGCGG-3’
Putative hemagglutinin
gene specific for subsp.
necrophorum
95°C for 5 min; 35
cycles of 95°C for 30
sec, 65°C for 30 sec,
and 72°C for 30 sec;
and final extension at
72°C for 4 min
311 1
Fund (for):
5’-CTCAATTTTTGTTGGAAGCGAG-3’
Fund (rev):
5’-CATTATCAAAATAACATATTTCTCAC-3’
Promoter region of
leukotoxin gene
specific for subsp.
funduliforme
94°C for 3 min; 35
cycles of 94°C for
1 min, 52.2°C for
30 sec, and 64.4°C
for 30 sec; 72°C
for 1 min; and final
extension at 72°C for
4 min
337 18
5’lktpxmxh (for):
5’-GAAATCTTTAAAGCAC-3’
3’lktpxmxh (rev):
5’-CATAATTTCTCCCAATTTTATT-3’
Promoter region of
leukotoxin gene in
subsp. necrophorum
and subsp. funduliforme
94°C for 3 min; 35
cycles of 94°C for
1 min, 52.2°C for
30 sec, and 64.4°C
for 30 sec; 72°C
for 1 min; and final
extension at 72°C for
4 min
571 in subsp.
necrophorum
and 449
in subsp.
funduliforme
18
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Fusobacterium necrophorum from llama and alpaca
505
strain. In 2 strains, 68690 and 68699, all bands were gener-
ally of low intensity. One strain (1012060) showed additional
high-intensity bands of 80, 60, and 40 kDa. Alpaca strains
61715 and 73556 showed 2 additional bands in the 30-kDa
range, and the 30-kDa band was more prominent. These 2
bands were absent in all other strains (Fig. 2). The higher
intensity of protein bands from strain 1012060 could be
either due to difference in the promotor sequence leading to
a higher expression of leukotoxin or to difference in amino
acid sequence of the protein increasing its affinity to the anti-
body. However, the higher expression of leukotoxin by the
strain 1012060 was not expected because the cytotoxicity of
this strain on polymorphonuclear leukocytes (PMNs) was
less compared to 2 other camelid strains (as described
below). In addition, the multiple bands that appeared in the
current analysis supports the previous observation that the
leukotoxin from F. necrophorum is highly unstable.
13,15
The
difference in the banding pattern among the strains is most
likely because of cleavage by proteolytic enzymes.
14
Like-
wise, the difference in intensities of similar bands in different
strains could also be attributed to the difference in the proteo-
lytic activity.
The leukotoxicity of the culture supernatant was assessed
by a cell viability assay using propidium iodide in a flow
cytometer.
10
For the assay, PMNs collected from the periph-
eral blood of cattle and alpaca were used.
14
The viable PMN
(1 × 10
6
) cells were treated with culture supernatants for 45
min at 37°C and 5% CO
2
, washed twice with phosphate buff-
ered saline (PBS), resuspended in PBS, and stained with 10
ml of propidium iodide (50 mg/ml) in the dark for 5 min. The
PMNs in complete Roswell Park Memorial Institute (RPMI-
1640
g
) medium and PMNs treated with supernatants auto-
claved (to denature the protein) from each strain served as
negative controls. The samples were processed on a flow
cytometer,
h
and data were analyzed using commercial
software.
i
The flow cytometry analysis showed that the
culture supernatants from llama and alpaca isolates were
cytotoxic to alpaca and bovine PMNs (Fig. 3). The statistical
analysis of cytotoxicity was performed using a generalized
linear model (PROC GENMOD).
j
For analyzing the cytotox-
icity of the culture supernatants of the isolates to alpaca and
bovine PMNs, a repeated measures analysis of variance
model with a serial correlation structure was used. The val-
ues were assumed to be normal, and a generalized linear
model was used to fit the model. Score statistics based on the
differences in the least square means were used to assess the
significance of strains, species, and their interactions. Results
were considered significant at P < 0.05.
The extent of cytotoxicity with either alpaca or bovine
PMNs differed among camelid strains. The cytotoxicity of
many of the camelid strains were higher (P < 0.05) with
alpaca PMNs compared to bovine PMNs. Similarly, strain
A25 (subsp. necrophorum) was more cytotoxic to bovine
PMNs compared to alpaca PMNs. However, the cytotoxicity
of the B35 strain (subsp. funduliforme) did not differ between
alpaca and bovine PMNs. Also, the 2 bovine strains did not
differ significantly in cytotoxicity with alpaca PMNs, but
strain A25 was more cytotoxic than strain B35 with bovine
PMNs. With alpaca PMNs, the cytotoxicities of 7 out of 9
camelid strains were higher than either of the 2 bovine
strains, cytotoxicity of 1 strain (118610) was similar to, and
that of another strain (69521) was lower than that of bovine
strains (Fig. 3). With bovine PMNs, the cytotoxicities of all
camelid strains, except strain 73556, were lower than that of
the bovine strain, A25 (Fig. 3). There was no difference in
the cell viabilities of PMNs treated with the autoclaved cul-
ture supernatant of either camelid or bovine strains with
PMNs suspended in RPMI-1640 as negative control (data
not shown), suggesting the toxin is a protein and not lipo-
polysaccharide.
The cell viability assay using alpaca and bovine PMNs
showed that the culture supernatants of camelid strains were
Figure 2. Western blot analysis of culture supernatants of Fusobacterium necrophorum strains from llama and alpaca. Lane M: protein
ladder; lane 1: bovine strain, A25 (positive control); lane 2: 11034; lane 3: 1012060; lane 4: 61557; lane 5: 61715; lane 6: 69521; lane 7:
68690; lane 8: 68699; lane 9: 73556; lane 10: 118610.
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Kumar et al.
506
cytotoxic to PMNs of both alpaca and bovine. However,
many of the strains of llama and alpaca isolates were more
toxic to the alpaca PMNs compared to bovine PMNs, which
may indicate some animal species specificity with regard to
cytotoxicity. The species specificity of bovine F. necropho-
rum leukotoxin has been previously reported.
4,15
The impor-
tance of leukotoxin as a virulence factor in fusobacterial
infections in camelids needs to be assessed. There is a sug-
gestion that leukotoxin is not a universal virulence factor of
those strains of F. necrophorum responsible for necrotic
infections in animals and human beings.
7
In the current study,
all the isolates from llama and alpaca were shown to have a
functional leukotoxin. It is possible that, in camelids, leuko-
toxin plays a major role in the establishment of necrotic
infections. In conclusion, the present study has characterized
Fusobacterium isolates of camelid origin. The Fusobacte-
rium isolates from the necrotic infections in alpaca and llama
belong to the subsp. necrophorum and the strains had leuko-
toxin activity, which could be a major virulence factor.
Acknowledgements
Mr. Sailesh Menon is thanked for his help and suggestions with
manuscript preparation and submission. Dr. Greg Peterson is also
thanked for laboratory support.
Sources and manufacturers
a. BD, Franklin Lakes, NJ.
b. RapID ANA II kit, Remel Inc., Lenexa, KS.
c. Eppendorf Mastercycler Gradient, Fisher Scientific, Pittsburgh, PA.
d. Bio-Rad Laboratories, Hercules, CA.
e. Sigma-Aldrich, St. Louis, MO.
f. Millipore Corp., Billerica, MA.
g. Life Technologies, Grand Island, NY.
h. FACSCalibur flow cytometer, BD Bioscience, San Jose, CA.
i. Cell Quest Analysis Software, BD, Franklin Lakes, NJ.
j. SAS v. 9.3, SAS Institute Inc., Cary, NC.
Declaration of conflicting interests
The author(s) declare no conflicts of interest with respect to the
research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: The
SUCCESS-FYI grant from the College of Veterinary Medicine at
Kansas State University partially supported the study.
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Figure 3. Cytotoxicity assay with culture supernatants of bovine and camelid strains of Fusobacterium necrophorum subsp.
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different (P < 0.05) from both bovine strains with alpaca PMNs (A) and from bovine strain A25 with bovine PMNs (B).
a
Cytotoxicity
with bovine PMNs significantly different (P < 0.05) from alpaca PMNs.
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Fusobacterium necrophorum from llama and alpaca
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... F. necrophorum is considered a normal inhabitant of the alimentary tract of alpacas. 8,11 Two subspecies of this microorganism have been identified: F. necrophorum ssp. necrophorum (biotype A) and F. necrophorum ssp. ...
... 12,13 In a study performed with F. necrophorum isolates obtained from different necrotic lesions in alpacas and llamas, leukotoxin was demonstrated in the culture supernatant in all isolates, suggesting that this toxin may play a role in the pathogenesis of necrotic lesions in these animal species. 11 In ruminants, most of the ulcerative lesions in the upper digestive system that are associated with F. necrophorum occur after physical damage to tissues (e.g., trauma, ischemia, and parasitic or viral infections), which provides an anaerobic environment. 12,23,25 Once established in the tissue, F. necrophorum proliferates, causing extensive foci of coagulative necrosis. ...
... T. pyogenes and F. necrophorum are normally present on mucous membranes of the alimentary tract of camelids. 8,11 Pseudomonas spp. have been implicated in opportunistic infections and linked to environmental sources, particularly aqueous systems. ...
Article
Ulcers of the oral cavity, esophagus, and gastric compartments of South American camelids are uncommon. Multifocal-to-coalescing ulcers were identified in the oral cavity, esophagus, and/or gastric compartments of 5 alpacas submitted for postmortem examination. Fusobacterium necrophorum was isolated from the lesions in all alpacas, in combination with other aerobic and anaerobic bacteria. In 4 of these cases, F. necrophorum–associated lesions were considered secondary to neoplasia or other chronic debilitating conditions; in 1 case, the alimentary ulcers were considered the most significant autopsy finding. It is not known if this agent acted as a primary or opportunistic agent in mucosal membranes previously damaged by a traumatic event, chemical insult, immunodeficiency, or any other debilitating condition of the host.
... Necrobacteriosis is an infectious disease characterized by purulent-necrotic lesions localized mainly on the lower parts of the limbs, and in some cases in the oral cavity, udder, genitals, liver, lungs, muscles and other tissues and organs [6]. The causative agent Fusobacterium necrophorum, a Gram-negative anaerobic bacterium, is an opportunistic pathogen in animals and humans that causes various infections called necrobacteriosis [7,8]. F. necrophorum is ubiquitous in soil and manure, on the skin and hooves of domestic animals [9]. ...
... The causative agent Fusobacterium necrophorum, a Gram-negative anaerobic bacterium, is an opportunistic pathogen in animals and humans that causes various infections called necrobacteriosis [7,8]. F. necrophorum is ubiquitous in soil and manure, and on the skin and hooves of domestic animals [9]. ...
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Necrobacteriosis of cattle causes significant economic damage to the animal husbandary, which consists of a decrease in milk productivity, a decrease in fulness, lost weight and productivity and the cost of treating sick animals [1]. Annual studies show that in many farms of the republic, purulent - necrotic diseases of the limbs in cattle and small cattle are widespread. In some farms, especially in the northern and eastern regions of the republic, the incidence of cows with necrobacteriosis reaches 30–56% of the total population [2]. Necrobacteriosis is characterized by purulent-necrotic lesions of the skin, mucous membranes of the underlying tissues, sometimes parenchymal organs; while, as a rule, distal parts of the limbs are affected. This infection affects all types of domestic animals and most wild animals. Cattle are most susceptible, especially pregnant heifers and dairy cows. Young animals are much more sensitive than adult animals[5]. Necrobacteriosis is a disease of the hooves of cattle caused by the pathogen Fusobacterium necrophorum. This disease is quite common both on large and small farms, and the fight against it remains an urgent problem due to the unsystematic use of antibiotics, resistance of the pathogen to drugs occurs, immunity decreases, and the treatment of infection is a laborious process [2, 4]. Necrobacteriosis affects many animal species, affecting various body systems [1]. The diagnosis of the disease was made in a complex manner, taking into account clinical signs and laboratory results. According to the results obtained, the isolated cultures, two isolates, according to morphological, cultural-physiological and biochemical properties, are attributed to the pathogen Fusobacterium necrophorum. The affiliation of the isolated pure culture of necrobacteriosis was determined by the determinant of bacteria of Bergi (1997).As a result, it was found that the causative agent of necrobacteriosis and the accompanying microflora were the most sensitive to tetracycline.
... Fusobacterium necrophorum was the species with the highest abundance in the microbiota of deer with dental abscesses, especially in the microbiota of a young marsh deer. This microorganism has already been identified in different species of deer with mandibular abscess and necrobacillosis (Roeder et al. 1989, Chirino-Trejo et al. 2003, in llamas and alpacas with necrotic infection (Kumar et al. 2013) and in goats with periodontitis (Suzuki et al. 2006, Campello et al. 2019. The high prevalence of this microorganism and the reduced diversity of other species identified in the young marsh deer may be related to the immaturity of the oral microbiota, as it was a 5-month-old cervid. ...
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Dental abscess in ruminants is an acute polymicrobial infection, usually resulting from periodontal disease or endodontic infection, with consequences for animal health and welfare. The present study aimed to describe the bacterial microbiota of dental abscesses in Blastocerus dichotomus. Biological material from mandibular dental abscesses, punctured with a sterile syringe and needle during routine veterinary curative procedures or necropsies, was collected from three ex-situ marsh deer. Bacteria were identified using high-throughput sequencing of the 16S ribosomal RNA gene. The three specimens had the presence of facial bulging, and two died because of severe emaciation with a history of progressive weight loss. Bacteroides (38.6%), Fusobacterium (36.65%), and Porphyromonas (7.49%) represented the most abundant genera and Fusobacterium necrophorum (35.69%), Porphyromonas levii (3.12%) and Porphyromonas gulae (1.78%) were among the ten most represented species in the microbiota of mandibular abscess in Blastocerus dichotomus. These molecular findings demonstrate a broader diversity of species in the polymicrobial nature of dental abscesses in B. dichotomus than was previously reported when culture-dependent methods were used in the diagnosis.
... Fusobacterium necrophorum is one of the common anaerobic bacteria that can cause abscesses and respiratory infections in animals [21]. Common diseases caused in commercial animals are liver abscess [22], endometritis [23] and foot rot [24] in cattle and sheep, while Fusobacterium necrophorum has also been reported in wild animals such as North American bighorn sheep [25], white-tailed deer [26,27], tundra caribou and alpacas [28]. This study is the first to discover forest musk deer abscess disease caused by Fusobacterium necrophorum as the dominant pathogen. ...
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Simple Summary Abscess disease is a major disease that affects forest musk deer populations. Accurately identifying the types of pathogenic bacteria responsible for it is crucial for effective clinical treatment and the development of drugs and vaccines. This study is the first to use high-throughput 16S rRNA sequencing technology to detect the types and abundance of pathogenic bacteria in abscess disease samples at the genetic level, thereby overcoming limitations of previous methods. Microbial structure and bacterial community correlation analyses of six sequencing samples revealed that the dominant pathogenic bacteria were relatively singular and had an overwhelming abundance in the same individual. The pathogenic bacterial species differed among different individuals, and the dominant pathogenic bacteria exhibited no significant correlation with other bacteria in the pus, thus indicating that the dominant pathogenic bacteria were responsible for the production of abscess disease. The primary dominant pathogenic bacteria were Trueperella pyogenes, Fusobacterium necrophorum, and Bacteroides fragilis. While Trueperella pyogenes has been confirmed as one of the pathogenic bacteria responsible for abscess disease in forest musk deer, Fusobacterium necrophorum and Bacteroides fragilis could not be isolated or identified by previous research methods due to their obligate anaerobic characteristics. Therefore, this study is the first to report that Fusobacterium necrophorum and Bacteroides fragilis are the dominant pathogenic bacteria responsible for abscess disease in forest musk deer. Abstract Currently, researchers use bacterial culture and targeted PCR methods to classify, culture, and identify the pathogens causing abscess diseases. However, this method is limited by factors such as the type of culture medium and culture conditions, making it challenging to screen and proliferate many bacteria effectively. Fortunately, with the development of high-throughput sequencing technology, pathogen identification at the genetic level has become possible. Not only can this approach overcome the limitations of bacterial culture, but it can also accurately identify the types and relative abundance of pathogens. In this study, we used high-throughput sequencing of 16S rRNA to identify the pathogens in purulent fluid samples. Our results not only confirmed the presence of the main pathogen reported by previous researchers, Trueperella pyogenes, but also other obligate anaerobes, Fusobacterium necrophorum and Bacteroides fragilis as the dominant pathogens causing abscess diseases for the first time. Therefore, our findings suggest that high-throughput sequencing technology has the potential to replace traditional bacterial culture and targeted PCR methods.
... funduliforme was isolated from clinical human infections and their virulence is determined by secreting leukotoxin. In humans, F. necrophorum is responsible for Lemierre syndrome, which begins as bacterial pharyngitis and rapidly progresses to septic thrombophlebitis of the jugular vein [87][88][89][90]. Fusobacterium nucleatum was enriched in colorectal carcinoma. ...
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Background The microbiome alterations are associated with cancer growth and may influence the immune system and response to therapy. Particularly, the gut microbiome has been recently shown to modulate response to melanoma immunotherapy. However, the role of the skin microbiome has not been well explored in the skin tumour microenvironment and the link between the gut microbiome and skin microbiome has not been investigated in melanoma progression. Therefore, the aim of the present study was to examine associations between dysbiosis in the skin and gut microbiome and the melanoma growth using MeLiM porcine model of melanoma progression and spontaneous regression. Results Parallel analysis of cutaneous microbiota and faecal microbiota of the same individuals was performed in 8 to 12 weeks old MeLiM piglets. The bacterial composition of samples was analysed by high throughput sequencing of the V4-V5 region of the 16S rRNA gene. A significant difference in microbiome diversity and richness between melanoma tissue and healthy skin and between the faecal microbiome of MeLiM piglets and control piglets were observed. Both Principal Coordinate Analysis and Non-metric multidimensional scaling revealed dissimilarities between different bacterial communities. Linear discriminant analysis effect size at the genus level determined different potential biomarkers in multiple bacterial communities. Lactobacillus, Clostridium sensu stricto 1 and Corynebacterium 1 were the most discriminately higher genera in the healthy skin microbiome, while Fusobacterium, Trueperella, Staphylococcus, Streptococcus and Bacteroides were discriminately abundant in melanoma tissue microbiome. Bacteroides, Fusobacterium and Escherichia-Shigella were associated with the faecal microbiota of MeLiM piglets. Potential functional pathways analysis based on the KEGG database indicated significant differences in the predicted profile metabolisms between the healthy skin microbiome and melanoma tissue microbiome. The faecal microbiome of MeLiM piglets was enriched by genes related to membrane transports pathways allowing for the increase of intestinal permeability and alteration of the intestinal mucosal barrier. Conclusion The associations between melanoma progression and dysbiosis in the skin microbiome as well as dysbiosis in the gut microbiome were identified. Results provide promising information for further studies on the local skin and gut microbiome involvement in melanoma progression and may support the development of new therapeutic approaches.
... Laryngeal lesions, comparable to those described in sheep, have been reported in calves [15], camelids [16], a white-tailed deer (Odocoileus virginianus) [17], thoroughbreds [18] and humans [19]. In calves, similar lesions are part of oral necrobacillosis, which is caused by a monoinfection with F. necrophorum [15]. ...
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Background: Ovine laryngeal chondritis is a rare entity of sheep in the USA, Great Britain, New Zealand and Iceland, but has not been reported in Germany so far. Here, two German cases are reported. Case presentation: Two rams showed severe and progressive signs of dyspnea. Endoscopically, a severe bilateral swelling of the larynx was identified in both rams. Due to poor prognosis and progression of clinical signs one ram was euthanized, while the other ram died overnight. In both cases, a necrosuppurative laryngitis and chondritis of arytenoid cartilages was found at necropsy. Fusobacterium necrophorum and Streptococcus ovis were isolated from the laryngeal lesion in one animal. Conclusions: This is the first report of ovine laryngeal chondritis in continental Europe. This entity should be considered a differential diagnosis for upper airway disease in sheep.
... 27 Amplification by PCR of the lkt promoter and the hemagglutinin gene differentiated F. necrophorum subspecies from other Fusobacterium spp. 10 In the current study, the inconsistency in the band produced by F. n. subsp. funduliforme with fund primers may represent polymorphisms in the sequence of this virulence gene. ...
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A total of 23 clinical isolates of Fusobacterium spp. were recovered at necropsy over a 2-year period from the respiratory tract of white-tailed deer (Odocoileus virginianus). Isolates were identified as Fusobacterium varium (18/23), Fusobacterium necrophorum subsp. funduliforme (3/23), and Fusobacterium necrophorum subsp. necrophorum (2/23). Using polymerase chain reaction-based detection of virulence genes, all F. necrophorum isolates were positive for the promoter region of the leukotoxin operon and the hemagglutinin-related protein gene, while all F. varium isolates were negative. The presence of the leukotoxin gene in F. necrophorum isolates and the absence of this gene in F. varium isolates were confirmed by Southern hybridization using 2 separate probes. Toxicity to bovine polymorphonuclear leukocytes was observed with all F. necrophorum isolates, but was not observed in any F. varium isolates. Susceptibility to antimicrobials was markedly different for F. varium as compared to F. necrophorum. In summary, no evidence of leukotoxin production was detected in any of the 23 F. varium isolates used in the current study. The data suggests that F. varium, the most common species isolated, may be a significant pathogen in deer with a different virulence mechanism than F. necrophorum.
Chapter
This chapter highlights clinical features, differentials, and diagnostic information for bacterial skin diseases of camelid. Those diseases are folliculitis and sfurunculosis, corynebacterium pseudotuberculosis infection, dermatophilosis, pododermatitis, and miscellaneous bacterial diseases abscess. Folliculitis (hair follicle inflammation) and furunculosis (hair follicle rupture) are common and cosmopolitan. Cultural evaluations have not been reported, but anecdotal literature suggests that Staphylococcus aureus and S. intermedius are causative. Predisposing factors include trauma and moisture. C. pseudotuberculosis infection is uncommon and cosmopolitan. The organism contaminates various wounds, and moisture and flies are important contributing factors. Dermatophilus congolensis proliferates under the influence of moisture (especially rain) and skin damage. Pododermatitis is a multifactorial disorder seen in alpacas and llamas. Recognized causes include trauma, bacterial folliculitis, yeast infection, chorioptic mange, sarcoptic mange, contact dermatitis, insect-bite hypersensitivity, and zinc-responsive dermatitis.
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Anaerobic and aerobic cultures of facial and mandibular abscesses were made from 12 blue duiker (Cephalophus monticola fusicolor) housed at the Deer and Duiker Research Facility of the Pennsylvania State University (USA). Increases in concentrations of total protein and serum globulin occurred in all cases. Actinomyces pyogenes was isolated from nine animals. Fusobacterium necrophorum was present in eight and Bacteroides sp. was found in seven animals; other genera of isolated bacteria included: Streptococcus (from two animals), Lactobacillus (one), Staphylococcus (one) and Actinomyces (two). Eight (67%) of affected animals were less than or equal to 2 yr of age. Facial soft tissues and mandibles were the tissues most often affected. Tissues within the oral cavity were not affected at the time of presentation. A common finding, not reported in other host species with necrobacillosis, was the presence of nondestructive mandibular proliferation.
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Fusobacterium necrophorum is recognized as the cause of a severe life-threatening illness characterized by bacteraemia with metastatic abscesses following an acute sore throat (Lemierre's disease). However, the importance of F. necrophorum as a cause of simple sore throat in the community is unknown. Using quantitative real-time PCR with primers targeting the rpoB gene, 100 routine throat swabs collected from patients presenting to general practitioners with pharyngitis were analysed for the presence of F. necrophorum-specific DNA. The results were compared with those obtained from throat swabs collected from 100 healthy subjects. Ten clinical samples were positive for F. necrophorum DNA, identified as F. necrophorum subspecies funduliforme, using a haemagglutinin-related protein gene-specific PCR assay. All the healthy controls were negative (two-tailed P value = 0.0015; Fisher exact test). These findings suggest that F. necrophorum may play a more important role as a cause of simple sore throat in the community than has been previously appreciated.
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Necrobacillosis was diagnosed in 17 of 18 white tailed deer, nine of 17 mule deer and two of three pronghorn antelope found dead on a ranch in southwestern Saskatchewan during the period February to April 1974. Diagnosis was based upon gross and histopathological lesions, and the morphology of organisms present in impression smears of lesions. At least 40 deer and 17 antelope died in the area, but no disease was reported among approximately 600 sheep and 200 cattle sharing the range. History, field observations and pathology of the disease are described, and the epizootiology is discussed.
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The effect of cultural conditions on the production of leukotoxin by biotypes A and B of F. necrophorum was investigated. Biotypes A and B were grown in prereduced, anaerobically sterilized, brain-heart infusion (BHI) broth. The average leukotoxin titer of culture supernatant was 18 times higher from biotype A strains than from biotype B strains. Leukotoxin activity peaked during the late-log and early-stationary phases of growth, then declined precipitously in both biotypes. F. necrophorum biotype A was grown in different media (BHI, liver infusion, and Eugon broths), at various pH (6.6, 7.3, 7.7, and 8.2), incubation temperatures (30, 35, 39, and 43 degrees C), redox potentials (-352 to +375 mV), and iron concentrations (less than 0.2, 4.2, 42.1, and 361.4 microM). Anaerobic BHI broth with pH from 6.6 to 7.7 at 39 degrees C incubation temperature supported maximal F. necrophorum growth and leukotoxin production. The optimum redox potential for F. necrophorum growth was in the range of -230 to -280 mV. However, the presence of titanium III citrate or dithiothreitol (7.78 mM) in the medium decreased (P less than 0.05) the leukotoxicity of F. necrophorum. Low iron concentration (less than 0.2 microM) decreased (P less than 0.05) growth rate but not leukotoxin activity of F. necrophorum, whereas high iron concentration inhibited the leukotoxin activity.
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The biological and biochemical properties, DNA base compositions, and levels of DNA-DNA homology of two biovars of Fusobacterium necrophorum were examined. Some differences were found between the two biovars in biological and biochemical properties. The G + C contents of DNAs from biovar A strains VPI 2891T (T = type strain), NCTC 10576, N167, Fn47, and Fn43, were 32, 30, 29, 28, and 31 mol%, respectively. The G + C contents of DNAs from biovar B strains Fn524T, 606, Fn49, Fn45, and 1260 were 30, 31, 27, 31, and 30 mol%, respectively. Labeled DNA from biovar A strain VPI 2891T exhibited 100 to 80% relatedness to DNAs from biovar A strains and 59 to 51% relatedness to DNAs from biovar B strains. Labeled DNA from biovar B strain Fn524T exhibited 100 to 81% relatedness to DNAs from biovar B strains and 71 to 60% relatedness to DNAs from biovar A strains. Therefore, the names Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flügge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Hallé 1898), are proposed for Fusobacterium necrophorum biovars A and B, respectively. The type strain of F. necrophorum subsp. necrophorum is strain VPI 2891 (= JCM 3718 = ATCC 25286), and the type strain of F. necrophorum subsp. funduliforme is strain Fn524 (= JCM 3724).
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Twenty-eight abscessed livers were collected from feedlot cattle at an abattoir; specimens were obtained from 49 abscesses for bacteriologic culture and for histologic examination. Cultural procedures included techniques to enumerate and isolate facultative and obligate anaerobic bacteria. Anaerobic bacteria were isolated from all 49 abscesses, whereas facultative bacteria were isolated from only 22. Mean bacterial counts for anaerobic and facultative bacteria were 3 X 10(8) and 8 X 10(8) bacteria/g of purulent material, respectively. Fusobacterium necrophorum, the only anaerobe isolated, was detected in 100% of the abscesses. Fusobacterium necrophorum biotype A was isolated from 57% of the abscesses (in pure culture from 75%), and F necrophorum biotype B was isolated from 47% of the abscesses (from 96% with mixed infections). Corynebacterium pyogenes was the predominant facultative bacterium isolated. Histologic changes in abscesses were qualitatively similar; abscesses were pyogranulomatous, with a necrotic center surrounded by zones of inflammatory tissue. However, the severity of lesions varied, depending on the F necrophorum biotype involved. Portal triad fibrosis and bile-duct proliferation were most severe in biotype A and mixed biotype B infections and less severe in abscesses from which biotype B was isolated in pure culture.